There has recently been a surge of interest in the value and clinical potential of proteomic biomarkers (1). A general belief in the medical community is that the earlier a disease is treated, the more successful the therapeutic outcome (2). Consequently, the routine clinical availability of biomarker tests specific for early-stage neoplastic diseases has tremendous potential to dramatically improve public health, even using currently utilized therapeutic modalities. For example, clinical oncologists expect that biomarker detection of pre-metastatic solid tumors of the breast, lung, ovary, and colon could lead to a significant improvement in survival (2). Unfortunately, despite the urgent clinical need, in the past ten years, for all disease categories combined, only a handful of novel biomarkers have graduated to routine clinical use (3). The slow biomarker pipeline persists despite considerable efforts within diagnostics research. The reasons for this failure stem from fundamental technical and biologic roadblocks spanning the biomarker development pipeline from biomarker identification and measurement to initial clinical validation. Two of these major roadblocks are:
Low Abundance: Disease-relevant biomarkers may exist in exceedingly low concentrations within a complex mixture of body fluid proteins containing high-abundance proteins such as albumin.
Instability: Immediately after the blood or other body fluid is collected (e.g. by venipuncture), degradation of proteins can occur, which is mediated by endogenous or exogenous proteinases.
Sensitive and specific biomarkers are expected to exist in very low abundance. There is a great need to develop novel methods for enriching the yield of rare candidate biomarkers present in the small volumes of blood available in clinical study sets. Candidate biomarkers are expected to exist in very low concentrations and must be separated from high-abundance blood proteins, such as albumin, which exist in a billion-fold excess. Early-stage disease lesions such as premalignant cancer may arise within a tissue volume of less than 0.10 mL. Assuming all the putative biomarkers emanating from this volume are uniformly dispersed within the entire blood volume of 5,000 mL, then the dilution factor will be 50,000. One can also reasonably hypothesize that the most physiologically relevant proteins specific for the disease may constitute a minor subpopulation of the cellular proteome. Consequently, the greatest challenge to biomarker discovery is the isolation of very rare candidate proteins within a highly concentrated complex mixture of blood proteins massively dominated by non-relevant proteins. Thus, analytical sensitivity is the first challenge for biomarker discovery and measurement. The problem of low abundance protein detection extends from discovery to routine measurement. During the discovery phase, it is likely that large plasma or serum volumes, including pooled samples, can be available for analysis. In contrast, once a candidate marker is taken forward to clinical testing, the volume of blood available for an individual patient's assay may be less than 1.0 mL. Taking all of these factors into consideration, the analytical platform used to measure the candidate marker must have a detection sensitivity sufficient to reliably detect marker concentrations in the sub-femtomolar or attomolar concentration.
In addition, candidate blood biomarkers are highly perishable. Circulating protein biomarkers are subject to rapid proteolytic cleavage and modification immediately following blood procurement. The level of degradation depends on the manner of collection and the storage conditions immediately following venipuncture. For collection of serum, proteins are subject to cleavage by active enzymes in the clotting cascade. Depending on the time and temperature of incubation during clotting and the temperature of subsequent storage of the separated serum, the constellation of proteolytic fragments can be quite variable (4). If plasma is collected, clotting enzyme activity is suppressed, but endogenous proteinases may still be active. Moreover, depending on the means of plasma stabilization (e.g., heparin versus citrate), there can be significant chemical modifications of plasma proteins. Questionable stability of plasma or serum proteomics markers has been a major cause of perceived bias during the clinical validation of candidate protein biomarkers (5).